Receptors on liver cells play a fundamental role in normal liver growth and development, and function as targets for selective delivery of cDNA to hepatocytes (gene therapy). Their expression is tightly regulated; however, relatively little is known about the mechanisms responsible. To address these questions we propose to use as a model system the asialoglycoprotein (ASGP) receptor of hepatocytes which is expressed on the liver-derived cell line HepG2. It has been studied as a model of receptor-mediated endocytosis, a process which delivers to the cell nutrients, growth factors, pathogenic viruses and other proteins. In previous studies we identified a novel regulatory mechanism of receptor endocytosis: tyrosine kinase activation during the initial phase of receptor clustering and internalization by the cell. The proposed project will extend these findings to investigate several major questions. First, the role of tyrosine kinase activation in receptor endocytosis will be examined using intact and semi-intact cells. The cells used will be both liver-derived cells and other cell lines transfected with wild-type ASGP receptor cDNA and cDNA modified by site-directed mutagenesis. The semi- intact cells permit access of macromolecular probes such as antibodies to critical structural determinants of the receptor and the endocytic machinery. Second, the identity and regulation of cellular kinases which modulate receptor trafficking will be examined. Recently, we have identified a protein tyrosine kinase that is tightly associated with the ASGP receptor in a human hepatoma-derived cell line. This association is regulated in turn by a cellular GTP-binding protein. Studies to be performed include the characterization and identification of this tyrosine kinase, and the structural analysis of receptor-kinase interaction. Third, to investigate ASGP receptor function in normal liver development, specimens of human liver tissue obtained from the University of Minnesota and Nebraska Transplant Centers will be analyzed. As complex formation between ASGP receptor and kinase may modulate receptor function (described above), the abundance and activity of receptor kinase complexes will be measured in normal liver and compared to HepG2. The role of receptor-kinase association is completely unknown. The goal of these studies is an understanding of both the basic cell biology and the physiologic significance of this interaction.